Identification Of Myostatin As The Mh Locus In Cattle

One example where molecular genetics identified causal mutations of an extreme phenotype is the elucidation of double-muscling in cattle.[1] For nearly 200 years, the muscular hypertrophy (mh) syndrome called double-

nt419 del7-ins10

Q204X

C313Y

E226X

E291X

nt821 del11

NHoH

Propeptide

Fig. 1 Diagram of myostatin mutations that cause double muscling in cattle. Abbreviations for the amino acid change and the position in the coding region are shown by arrows. Six of the known mutations are 1) Q204X, which changes a glutamine to a termination signal; 2) E226X and 3) E291X, which change a glutamic acid to a stop codon; 4) nucleotide 419 in exon 2, deletion of 7, and insertion of 10 nucleotides (nt419del7ins10);

5) nt821deletion11 in exon 3 of Belgian Blue cattle, which alters the coding sequence and results in premature stop codons; and

6) C313Y, which changes a cysteine to a tyrosine residue in Piedmontese cattle, changing the coding sequence and again resulting in a premature stop codon. A model showing location of domain structures is shown in Fig. 2. (Adapted from Ref. 1.). (View this art in color at www.dekker.com.)

musculature has captured the attention of geneticists and livestock breeders. Affected cattle exhibit bulging musculature of the shoulders and hindquarters and are extremely efficient in production of lean, tender meat. A major drawback to this phenotype is higher birth weights and a consequent increase of dystocia, frequently requiring veterinary assistance during calving. A single autosomal recessive pattern of inheritance is characteristic of the phenotype, and ''carrier'' animals are intermediate in growth and body composition. The genetic map was used to show that the locus lies at the centromeric end of bovine chromosome 2.[2] In 1997, a group at John Hopkins University investigating members of the TGF-b family of growth factors discovered that targeted gene-knockout of myostatin (GDF-8) in mice led to a dramatic muscle-specific growth similar to that of double-muscled cattle.[3] Researchers then independently determined that myostatin mapped to the mh locus[4] and identified nucleotide changes in Belgian Blue and Asturiana de los Valles cattle that effectively ''knockout'' or cause loss of function mutations of the myostatin gene.[5] A surprisingly large number of different allelic forms of myostatin exist in several breeds of cattle, and body composition varies by individual, breed, and sex.[1,5] Six disruptive mutations have been discovered in this relatively small gene and several other polymorphisms exist that do not change the amino acid code or have an apparent affect on the function of the gene or phenotype (Fig. 1). Although the defect in myostatin was first presumed to have a common origin and mutation, it is now thought that this is not the case, since several haplotypes have been identified.1-5-1 Now that specific allelic variants have been characterized, efforts to select and produce animals with highly desirable phenotypes, i.e., greater yields of leaner meat and reduced dystocia, can be implemented by breeders. The discovery of mutations in myostatin that cause double-muscling was the first successful identification of a gene causing an extreme and economically exploitable phenotype in cattle.

CLONING THE RN" LOCUS IN PIGS

The first successful demonstration of positional cloning in a farm animal was the discovery of the Rendement Napole (RN") allele in Hampshire pigs. ''Positional cloning'' refers to identification of a mutation with no knowledge other than its approximate position in the genome. The RN (a measure of cooked weight to fresh weight) locus contains a dominant mutation with large effects on meat quality and processing yield. Affected animals have low ultimate muscle pH 24 hours after slaughter, reduced water-holding capacity, and reduced yield of cured, cooked product.[6] These effects are caused by a large

Markers

Restriction Sites 1 i i li

Centromere

Telomere

BACs

Telomere

BACs rggc gtc c[G/A]a gcg gca rggc gtc c[G/A]a gcg gca

Fig. 2 Diagram of the strategy used to positionally clone the mutation for Rendement Napole (RN"). Using DNA markers (red vertical lines), a contiguous alignment of large insert genomic clones called Bacterial Artificial Chromosomes (BACs; purple horizontal bars) were identified that cover the genomic region where the mutation most likely resided. These BACs were positioned by markers they contained and by a restriction enzyme map of the individual BACs (restriction sites shown as blue vertical lines). The gene responsible, PKRAG3 (green arrow), was identified in two overlapping BACs by position between the two flanking markers (red arrows) closest to the RN" mutation. The exonic organization of the gene and the position and sequence of the mutation is shown below the genome. The ''G'' (green) is the normal allele and the ''A'' (red) is the mutated allele that causes the sequence to code for a glutamine instead of arginine. (Adapted from Ref. [7].) (View this art in color at www.dekker.com.)

Q204X

nt821 del11

Fig. 1 Diagram of myostatin mutations that cause double muscling in cattle. Abbreviations for the amino acid change and the position in the coding region are shown by arrows. Six of the known mutations are 1) Q204X, which changes a glutamine to a termination signal; 2) E226X and 3) E291X, which change a glutamic acid to a stop codon; 4) nucleotide 419 in exon 2, deletion of 7, and insertion of 10 nucleotides (nt419del7ins10);

5) nt821deletion11 in exon 3 of Belgian Blue cattle, which alters the coding sequence and results in premature stop codons; and

6) C313Y, which changes a cysteine to a tyrosine residue in Piedmontese cattle, changing the coding sequence and again resulting in a premature stop codon. A model showing location of domain structures is shown in Fig. 2. (Adapted from Ref. 1.). (View this art in color at www.dekker.com.)

70%) increase in muscle glycogen without other pathological effects. The RN~ allele has been found only in Hampshire pigs and probably increased in frequency due to favorable effects on growth rate and meat content of the carcass. The RN~ mutation was mapped to porcine chromosome 15, and the pig/human comparative map indicated the corresponding human gene that lies on chromosome 2.[6] The discovery of the specific underlying mutation used the arduous approach of constructing a complete physical map of the genomic region by screening a large-insert swine genomic library for clones carrying genes that map to the target region of human chromosome 2 (Fig. 2). New probes were designed from these clones to rescreen the library and develop a series of overlapping clones that span the region containing the RN~ mutation. This ''contig'' of clones spanned over 2 million base pairs and was used to generate genetic markers to narrow the position of the mutation and identify clones that most likely contained the gene. These clones were sequenced to reveal the gene content, which resulted in matches to three known RNA transcripts. Only one of these transcripts, AMP-activated protein kinase (AMPK) g-subunit (PRKAG), appeared to be a reasonable candidate for RN~ effects.[7] AMPK is composed of three subunits: a catalytic a-subunit and 2 regulatory subunits, p and g. AMPK is activated by an increase in AMP, stimulates ATP-producing pathways, and inactivates glycogen synthase, the key regulatory enzyme of glycogen synthesis. Complete sequencing of the cDNA of this gene determined it was a novel AMPK g-subunit designated PRKAG3.[7] Screening of several rn+ and RN~ pigs of different breeds revealed that a mutation in a functional domain of the protein (Fig. 2) was exclusively associated with RN~, but not normal rn+ animals from Hampshire or other breeds, consistent with the idea that RN~ originated with the Hampshire breed. Since the discovery of the RN~ mutation in the PRKAG3 gene, other polymorphisms have been identified in PRKAG3 in commercial lines, some of which are associated with glycogen content and meat quality. This is another example where additional alleles of genes involved in major mutations have a significant affect on quantitative trait variation in livestock.

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